The E o' of cytochrome c on a self-assembled monolayer (SAM) modified gold electrode increased by 300 mV immediately on immersion in methanol. On re-immersion of the electrode in aqueous buffer the original faradaic response was restored, but over a period of 120 min, indicating that methanol causes a 10 significant change in conformation/orientation of the protein.Much effort has been devoted to understanding the electrochemical properties of redox proteins and enzymes. 1 For applications such as biosensors and biofuel cells, 2 it is desirable to have direct and fast electron transfer kinetics 15 between the electrode and the biological component of interest. 3 However, the rate of direct electron transfer (DET) may be inherently slow due to a range of factors including: inaccessibility of the electroactive group in the protein structure; adsorption of the protein in a manner which does 20 not promote fast electron transfer or denaturation of the protein. 4,5 Modified electrodes can enhance the rate of electron transfer, and provide the opportunity to conduct detailed thermodynamic and kinetic studies. 6,7 The use of selfassembled monolayers on electrodes to promote electron 25 transfer between the redox protein of interest and the electrode has been widely used. [7][8][9][10][11] Factors that influence the E o' of haem proteins include the nature of the axial ligands; the polarity of the haem environment and the extent of haem surface exposure. 12-15 30 Despite numerous studies, the full range and effects of the factors that contribute to E o' and their relative contribution has not yet been determined. While the majority of enzyme use occurs in aqueous media e.g. glucose analysis 16 , enzymes can also catalyse reactions in nonaqueous media 17 and can be used 35 in synthetic reactions. 18 However, there have been relatively few studies of the electrochemical properties of enzymes in organic media. Cyt c is one of the most extensively studied redox proteins 19 and has been widely used as a model for larger, more complex systems. 20 The reduction of the 40 immobilised haem fragment of cytochrome c, microperoxidase-11, has been examined in aqueous buffer and glycerol. The increase of 30 mV in E o' in glycerol was dominated by entropic changes. 21 Immobilised cytochrome c showed a smaller increase (7 mV) in E o' on immersion in 45 glycerol, however the change was dominated by enthalpic changes. 22 Faradaic responses were observed for MP-11 in acetonitrile and ethanol, with increases of 60 and 64 mV in E o' , respectively. 23 The reduction of cyt c in solution was examined in a range of mixed solvents, with reversible 50 responses (E o' ranging from 199 -274 mV vs SHE) obtained in methanol, acetonitrile, DMF and DMSO. 24-25 Here we describe the direct electrochemical response of cyt c in methanol at a SAM modified gold electrode. The E o' increased immediately on immersion in methanol, returning to the 55 original value in aqueous solution, but on a much longer time scale, indicating that methanol causes a si...
A multi component assembly consisting of the redox protein cytochrome c (cyt c) immobilised onto vertically aligned gold tipped semiconductor nanorods is described. Cyt c was 10 successfully immobilised using a thiol linker. A faradaic response demonstrated that the protein is electroactive in this ultra high density array. Fig. 1a and Fig. 1 b show high resolution scanning electron microscopy (HRSEM) and high resulution transmission electron microscopy (HRTEM) images of the inorganic component in the assembly. The SEM side view image (Fig. 1a) shows the vertically aligned nanorods which were 45 electophoretically deposited onto large 2 cm × 1 cm ITO coated glass substrates, (see ESI †, Fig. S1 and S2). Nanorods of 100 nm in length were obtained by controlled oriented attachement of as synthesised nanorods 35 nm in length. 7 The nanorods are bound to the substrate at the end facets and are 50 separated from each other (~ 2 nm) by interdigitated longchain octadecyl phosphonic acid ligands. The resulting perpendicularly aligned nanorods were spun cast with a solution of gold chloride to grow a single gold tip on each rod in the array (Fig. 1b and ESI †, Fig. S3). The procedure was optimized to form individual gold tips of 3 nm in size, on 60
Changes in the secondary structure of cytochrome c on immersion in methanol were monitored using circular dichroism. These changes occurred immediately in methanol, upon re-immersion in aqueous buffer, the secondary structure was restored in ca. 45 min. This method enables the secondary structure of proteins in non-aqueous solvents to be monitored in real time.
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